The virion host shutoff protein (vhs) of herpes simplex virus triggers accelerated degradation of cellular and viral mRNAs while sparing other cytoplasmic RNA species. Previous work has shown that vhs forms a complex with translation initiation factor eIF4H, which displays detectable RNase activity in the absence of other viral or host proteins. However, the contributions of eIF4H and other host factors to the activity and mRNA targeting properties of vhs have not yet been directly examined. An earlier report from our laboratory demonstrated that rabbit reticulocyte lysate (RRL) contains one or more factors that strongly stimulate the RNase activity of vhs produced in Saccharomyces cerevisiae. We report here that such yeast extracts display significant vhs-dependent RNase activity in the absence of mammalian factors. This activity differs from that displayed by vhs generated in RRL in that it is not targeted to the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). Activity was strongly enhanced by the addition of RRL, eIF4H, or the related translation factor eIF4B. RRL also reconstituted strong targeting to the EMCV IRES, resulting in a major change in the RNA cleavage pattern. In contrast, eIF4H and eIF4B did not reconstitute IRES-directed targeting. These data indicate that eIF4B and 4H stimulate the nuclease activity of vhs, and they provide evidence that additional mammalian factors are required for targeting to the EMCV IRES.Herpes simplex virus (HSV) is a large, enveloped DNA virus that establishes a latent infection in neural cells. During lytic infection, viral genes are expressed in a coordinately and temporally regulated fashion through both transcriptional and posttranscriptional controls (51). HSV genes fall into three kinetic classes: immediate early (IE or ␣), early (E or ␤), and late (L or ␥). The IE genes are transcribed in the absence of de novo protein synthesis and stimulate the expression of the E and L genes. The E genes encode proteins required for DNA synthesis, and the L genes encode structural proteins. The HSV virion contains a number of regulatory proteins that affect the earliest events of viral infection. These proteins are part of the tegument-an amorphous protein layer located between the capsid and the envelope. The tegument-associated regulatory proteins are delivered into the cytoplasm upon fusion of the virion envelope with the host cell membrane and can therefore act prior to the onset of viral gene expression. One of these regulatory proteins is the virion host shutoff (vhs) protein, encoded by the UL41 gene, which triggers rapid shutoff of host protein synthesis following HSV infection (12,13,19,42,45,46,55,57).Upon delivery into the host cytoplasm, the 58-kDa vhs protein directs the shutoff of host protein synthesis, disruption of preexisting polyribosomes, and the degradation of cellular mRNAs in the absence of de novo viral gene expression (11, 14-16, 27, 28, 40, 41, 46, 63). The destabilization of cellular mRNAs by vhs is thought to aid viral mRNAs ...

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells play key roles in limiting herpesvirus infections; consequently, many herpesviruses, including herpes simplex virus (HSV), have evolved diverse strategies to evade and/or disarm these killer lymphocytes. Previous studies have shown that CTL and NK cells are functionally inactivated following contact with HSV-infected fibroblasts. During studies of the mechanisms involved, we discovered that HSV-inactivated NK-92 NK cells and Jurkat T cells contain a strikingly prominent, novel, ca. 90-kDa tyrosine-phosphorylated protein that we identified as the HSV tegument protein VP11/12. Inasmuch as VP11/12 produced in fibroblasts and epithelial cells is not obviously tyrosine phosphorylated, these data suggested that VP11/12 serves as the substrate of a cell-type-specific protein tyrosine kinase. Consistent with this hypothesis, VP11/12 was also tyrosine phosphorylated in B lymphocytes, and this modification was severely reduced in Jurkat T cells lacking the lymphocyte-specific Src family kinase Lck. These findings demonstrate that HSV tegument proteins can be differentially modified depending on the cell type infected. Our data also raise the possibility that VP11/12 may modulate one or more lymphocyte-specific signaling pathways or serve another lymphocyte-specific function. However, HSV type 1 mutants lacking the UL46 gene retained the ability to block signaling through the T-cell receptor in Jurkat cells and remained competent to functionally inactivate the NK-92 NK cell line, indicating that VP11/12 is not essential for lymphocyte inactivation. Further studies are therefore required to determine the biological function of tyrosine-phosphorylated VP11/12.